A major activity in DNA replication is the unwinding of complementary DNA strands by a helicase to produce template for polymerase factors. In eukaryotes, the Minichromosome Maintenance complex (MCM2-7) forms the core of the replicative helicase; however, despite its essential role, fundamental aspects of how MCM2-7 functions are not understood. The proposed studies will determine the molecular mechanism for two critical events in the life cycle of the metazoan replicative helicase: recruitment to sites of initiation and activation of helicase activity. The strength of this proposal draws from both the fundamental nature of the questions being asked and the interdisciplinary approach that will be used to answer them. In Aim 1, I will employ Drosophila genetics and biochemistry to identify the mechanism that chaperones MCM2-7 into a stable complex with Cdt1, an essential helicase loading factor. Although the MCM2-7?Cdt1 complex is a known intermediate in the loading reaction, this complex has not been isolated from metazoans due to an unrecognized regulatory mechanism. I will define this mechanism, which will provide critical insights for future efforts aimed at fully reconstituting metazoan helicase loading in vitro. Following MCM2-7 recruitment and loading, the helicase is activated through stable association with two additional protein factors, Cdc45 and GINS (CMG complex). How Cdc45 and GINS activate the latent helicase activity of MCM2-7 is unknown. In Aim 2, I will use cryo-EM to determine the sub-nanometer structure of the CMG stalled in the midst of an unwinding cycle. This structure will provide a direct physical explanation for how Cdc45 and GINS remodel MCM2-7 and substrate DNA to facilitate helicase activation. The proposed work overall will have significant implications for th field of DNA replication, and will help elaborate the role these factors play in diseases such as cancer and dwarfism that result from replication initiation defects.